Sublethal β-lactam antibiotics induce PhpP phosphatase expression and StkP kinase phosphorylation in PBP-independent β-lactam antibiotic resistance of Streptococcus pneumoniae

Abstract

StkP and PhpP of Streptococcus pneumoniae have been confirmed to compose a signaling couple, in which the former is a serine/threonine (Ser/Thr) kinase while the latter was annotated as a phosphotase. StkP has been reported to be involved in penicillin-binding protein (PBP)-independent penicillin resistance of S. pneumoniae. However, the enzymatic characterization of PhpP and the role of PhpP in StkP-PhpP couple remain poorly understood. Here we showed that 1/4 minimal inhibitory concentration (MIC) of penicillin (PCN) or cefotaxime (CTX), the representatives of {beta}-lactam antibiotics, could induce the expression of stkP and phpP genes and phosphorylation of StkP in PCN/CTX-sensitive strain ATCC6306 and three isolates of S. pneumoniae (MICs: 0.02-0.5 g/ml). The product of phpP gene hydrolyzed PP2C type Ser/Thr phosphotase-specific RRA(pT)VA phosphopeptide substrate with the Km and Kcat values of 277.35 mol/L and 0.71 S-1, and the hydrolytic activity was blocked by sodium fluoride, a PP2C type Ser/Thr phosphatase inhibitor. The phosphorylation levels of StkP in the four phpP gene-knockout ({Delta}phpP) mutants were significantly higher than that in the wild-type strains. In particular, the MICs of PCN and CTX against the {Delta}phpP mutants were significantly elevated as 4-16 g/ml. Therefore, our findings confirmed that sublethal PCN and CTX act as environmental inducers to cause the increase of phpP and stkP gene expression and StkP phosphorylation. PhpP is a PP2C type Ser/Thr protein phosphatase responsible for dephosphorylation of StkP. Knockout of the phpP gene results in a high level of StkP phosphorylation and PBP-independent PCN/CTX resistance of S. pneumoniae.\n\nImportanceStreptococcus pneumoniae is a common pathogen in human populations in many countries and areas due to the prevalence of {beta}-lactam antibiotic-resistant pneumococcal strains. Production of {beta}-lactamases and mutation of penicillin-binding proteins (PBP) have been considered as the major {beta}-lactam antibiotic-resistant mechanisms in bacteria, but S. pneumoniae has not been confirmed to produce any {beta}-lactamases and many pneumococcal strains present PBP mutation-independent {beta}-lactam antibiotic resistance. StkP is a Ser/Thr kinase of S. pneumoniae to compose a signal-couple with PhpP protein. The present study demonstrated that the PhpP is a PP2C-type phosphotase for dephosphorylation of StkP and the sublethal penicillin (PCN) or cefotaxime (CTX) acted as environmental signal molecules to induce the expression of PhpP. The knockout of PhpP-encoding gene caused the PCN/CTX resistance generation of PCN/CTX-sensitive pneumococcal strains. All the data indicate that StkP-PhpP couple of S. pneumoniae is involved in PBP mutation-independent {beta}-lactam antibiotic resistance by phosphorylation of StkP.